Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined ‘vomocytosis’. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition—Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as ‘vomocytes’. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.


Introduction
Numerous pathogens have demonstrated means for persistence, dissemination, and infection within their mammalian hosts. The fungal species Cryptococcus neoformans (CN) is an opportunistic pathogen that causes an infectious disease called 'Cryptococcosis' that predominantly affects immunocompromised patients-primarily those afflicted with HIV/AIDS [1][2][3]. This infection may progress to a condition called Cryptococcal Meningitis (CM), where CN establishes an infection in the central nervous system (CNS). Globally, there are estimated to be 223,100 CM cases and 181,000 deaths annually [1]. Cryptococcus neoformans cells typically enter the host through the lung via inhalation. Subsequently, the fungal pathogen disseminates from the lung into other tissues, including the CNS [2,4]. The three proposed mechanisms that facilitate CN entry into the CNS are paracytosis, transcytosis, and hitchhiking. In the latter, CN are suggested to cross the blood brain barrier (BBB) by 'hitchhiking' within host phagocytes [5,6]. This is known as the 'Trojan Horse' hypothesis. Kechichian et al. showed that depletion of alveolar macrophages (MF) in mice significantly reduces cryptococcal dissemination to the CNS [7]. Charlier et al. later showed that infecting naïve hosts with CN-infected monocytes significantly increases CN accumulation in the brain, compared to infecting with CNs directly [8]. Recently, Gilbert et al. also linked CN dissemination (muscular and CNS) to length of time residing within phagocytes in an in vivo zebrafish model, supporting the Trojan Horse mechanism of CN transport [9].
Like most cell types, MFs regularly perform exocytosis to recycle membrane components and excrete various factors [13][14][15]. However, vomocytosis is a unique form of exocytosis whereby these immune cells (over 5 to 20 hours) expel large pathogenic particulates that they are otherwise programmed to be retained and digested. Studies have identified intracellular, physicochemical, and immunological cues in CN-infected MFs that are linked to this mechanism. As MFs perform vomocytosis, actin rapidly and transiently polymerizes in a cage-like structure around the phagosome, which then fuses with the plasma membrane [16]. These cages may be a post-phagosome permeabilization attempt by the MF to inhibit CNs' escape, as inhibiting actin polymerization has been shown to increase vomocytosis occurrence. In addition, CN disrupts phagolysosomal maturation, as characterized by the rapid removal of the early phagosome markers Rab5 and Rab11 [17]. Some reports have suggested that there is alkalinization of, and abnormal calcium ion levels in phagosomal compartments that contain the live pathogen [17][18][19][20][21]. Furthermore, the addition of weak bases to CN-infected MFs has been shown to modulate vomocytosis occurrence from MFs [18,21]. Moreover, Gilbert et al. showed that pharmacological inhibition of ERK5 increases vomocytosis occurrence [9].
Further, the immune state of infected MFs has been suggested to also influence this phenomenon. A recent study by Seoane et al. discovered that viral exposure to either measles or human immunodeficiency virus (HIV) were both shown to significantly boost expulsion rates of CN cells from MFs [22]. Moreover, other factors known to elicit an antiviral response-the TLR3 agonist poly(I�C) and type I interferons, IFN-α and IFN-β-all similarly increase MF vomocytosis events. Another study investigated how different T cell effector-induced phenotypes can impact the expulsion rates of infected J774 MFs [23]. Prior to infection, these cells were treated with cytokines inducing T effector cell-induced phenotypes Th1 (IFN-γ and TNF-α), Th2 (IL-4 and IL-13), or Th17 (IL-17). The Th1 and Th17 subtypes of J774 cells showed diminished intracellular CN proliferation and increased vomocytosis rates, whilst the Th2 group displayed increased intracellular CN proliferation and reduced vomocytosis occurrence.
Taken altogether, studies have given some clarity on the influence of intracellular, physicochemical, and immune states on vomocytosis. However, they focus entirely on MFs, which are only a single cell type in an army of immune cells. Moreover, Yang et al. recently discovered the occurrence of this phenomenon in neutrophils [24].
Like MFs and neutrophils, dendritic cells (DCs) have the unique ability to phagocytose particulates, including pathogens. More importantly, DCs link the innate and adaptive arms of the immune system. These innate immune cells are key for maintaining a balance between the host defense against pathogens and protection of "self" antigens of host cells and tissues [25][26][27]. Dendritic cells detect invading pathogens due to their constituent sensors (e.g. Toll-like receptors [TLRs]) [28,29]. They communicate the presence of pathogens to the adaptive immune system, thereby initiating long lasting, antigen-specific responses. Migration of DCs to T cell-rich regions is critical here and is mainly regulated by the chemokine receptor CCR7 [30][31][32] and CCL21 [33][34][35]. Following DC migration to secondary lymphoid organs, lymphocytes are subsequently activated and induced to proliferate and become potent effector cells (e.g. helper T cells) [25]. Interestingly, other innate immune cell types can traffick via lymphatics [32,36] and perform antigen presentation [37][38][39]. While these cell types have some promising capability in lymphatic migration and antigen presentation, their abilities are limited in comparison to DCs, which are recognized as the primary antigen presenting cell type whose dominant function is to traffick to the lymph nodes (LNs) to present foreign material to LNresident T cells.
During cryptococcal infections, murine, bone marrow-derived DCs are known interact with CN cells and have been demonstrated to phagocytose CN cells following opsonization with complement or antibody [40]. Hole et al. demonstrated the ability of murine, bone marrow-derived DC lysosomal extract to cause morphological changes in CN and kill the pathogen in vitro via oxidative and non-oxidative mechanisms [41]. Artavanis-Tsakonas et al. showed that CN-containing murine, bone marrow-derived DC phagosomes have an impaired CD63 recruitment, indicative of a distinct phagosomal compartment composition that may affect the outcome of antigen processing and presentation [42]. However, to the best of our knowledge, the ability of DCs to expel CN from their phagosome via vomocytosis has not been investigated thus far.
We hypothesized that DCs could perform vomocytosis, especially given that they share much of the same vacuolar machinery as their phagocytic relatives, MFs and neutrophils. In this present study, the DCs used are defined as GM-CSF differentiated cells from murine, bone marrow-derived progenitors and characterized by presence of CD11c and absence of F4/ 80 surface markers. Herein, we endeavored to document vomocytosis from DCs-a key player in bridging the innate and adaptive immune response. Further, we investigated the effect of CN infection ratio, presence of CN capsule, drug manipulation of the phagosome conditions and actin polymerization on vomocytosis from DCs. The overall effect of the immune state on vomocytosis from both DCs and MFs was also assessed. Finally, we characterized vomocytosis based on multiple outcomes-rate of vomocytosis occurrence, timing of expulsion, and number of internalized CN prior to expulsion. We believe that further investigation of CN's complex interactions with different phagocytic cell types will act as a step towards elucidating the complex story of cryptococcal infections and underlying mechanisms of vomocytosis.

Bone marrow-derived DC and MF culture
All animals were maintained and used in accordance with NIH guidelines and approved by UC Davis Institutional Animal Care and Use Committee (approval number 21840). Mice were housed four per cage, 20-26˚C ambient temperature, 12-hour light/dark cycle, with ad libitum access to food and water. Animals were monitored by husbandry staff at least once every day, with monthly healthcare checks by a veterinarian. Consistent with the recommendations of the Panel on Euthanasia of the AVMA, mice in this study were euthanized by lethal dose of CO 2 asphyxiation followed by cervical dislocation. This method of euthanasia is not painful and minimizes any discomfort experienced by the animal. Primary DCs and MFs were obtained from the bone marrow of C57BL/6 mice as described in previous studies [43][44][45]. Growth media consisted of DMEM/F-12 1:1 with L-glutamine (Cellgro, Herndon, VA), 10% fetal bovine serum, 1% sodium pyruvate (Lonza, Walkersville, MD), 1% nonessential amino acids (Lonza, Walkersville, MD), 1% penicillin/streptomycin (Cytiva, Marlborough, MA) and 20 ng/mL GM-CSF (R&D Systems, Minneapolis, MN) (DC media) or 10% L929 conditioned media (MF media) and incubated at 37˚C and 5% CO 2 . For conciseness, media containing all necessary growth factors and added reagents for the necessary cell type will be denoted as 'complete media'. Unless otherwise noted we used DC and MF on day 10 of their respective cultures.

Cell phenotype validation via RNA analysis
On day 10 or 11 of DC or MF culture, the contents of cells were extracted via application of TRIzol (Thermo Fisher Scientific, Waltham, MA). To isolate RNA from samples, a chloroform solvent extraction was performed according to manufacturer instructions. Next, RNA was purified using a kit, RNA Clean & Concentrator with DNAse (Zymo Research, Irvine, CA). The DNA Technologies Core at UC Davis assessed RNA quality (score>7.0), performed Batch 3'Tag-Seq library preparation, and sequenced using an Illumina NextSeq sequencer (Illumina, San Diego, CA). For analysis, reads were trimmed, aligned, and quantified for gene counts using OmicSoft software (Qiagen, Hilden, Germany). Dimensional reduction for principal component analysis (PCA) plots was also performed in the OmicSoft software.

Dendritic cell and MF polarization validation
Dendritic cells and MFs were seeded on 12 well plates (0.5 million cells/ well [DCs] and 0.25 million cells/ well [MFs]) in complete media containing polarizing agents. The difference in seeding density was due to spreading ability, with MFs being much more elongated than DCs and therefore taking up more surface area per cell. For inflammatory activation, cells were treated with LPS (100 ng/ml) from Escherichia coli O111:B4 (Sigma, St. Louis, MO). For antiinflammatory polarization DCs were treated with 1uM of dexamethasone (DEX; Alfa Aesar, Tweksbury, MA) and MFs with IL-4 (20ng/ml) and IL-13 (20ng/ml; denoted 'IL4/13' for brevity; R&D Systems) in MF media. After a 48-hour incubation period, the expressions of cell surface markers were determined via flow cytometry using antibodies against F4/80, CD38 Additionally, an LPS activation resistance test was performed by adding LPS (100 ng/ ml) to DCs and MFs previously treated with tolerogenic polarization agents for an additional 48 hours. Subsequently, the expressions of the same cell surface markers were quantified using flow cytometry.

Effect of infection rate on vomocytosis
Dendritic cells and MFs were seeded on 24 well plates (75,000 cells/ well [DCs] and 50,000 cells/ well [MFs]) in complete media and incubated at 37˚C and 5% CO 2 . Infections with CNs and subsequent time-lapse imaging studies were performed between day 11-15. Wildtype CN H99 and the acapsular mutant cap59 CN (both generously gifted by Dr. Angie Gelli, UC Davis, CA) were grown first on yeast extract peptone dextrose (YPD) agar (Thermo Fisher Scientific) followed by transferring a single colony to YPD broth (Thermo Fisher Scientific) shaking at 30˚C overnight. The next day, CNs were washed with PBS (x3) via centrifugation. The heat-killed (HK) CN negative control group was prepared by incubating CNs at 70˚C on a heat block for 1 hour. Both live and HK CNs were opsonized with 10 ug/ml of the anti-capsular IgG1 monoclonal antibody 18B7 (supplied from both Sigma and the Casadevall Lab, Johns Hopkins University, MD) and 50% human AB serum (Sigma). Opsonized pathogen was coincubated with phagocytes at a 1:1 or 5:1 CN:phagocyte ratio (c.p.r.) for 2 hours in media with 10% human AB serum. Next, infected culture wells were washed with complete media (x5) to ensure all extracellular CNs were removed. Lastly, complete media was added, and time-lapse imaging was performed on infected cells for 14 hours.

Effect of drugs on vomocytosis
For drug-treated experimental groups, DCs were infected with CN at a 5:1 c.p.r. using identical methods outlined previously. After the 2-hour phagocytosis and washing step, the wells were treated with DC media containing either 10 uM of chloroquine [CQ] (Thermo Fisher Scientific), 100 nM of cytochalasin B from Drechslera dematioidea [CYT low] (Sigma), 4 uM of cytochalasin B (CYT hi), or 100 nM of bafilomycin A1 [BFA] (Sigma). These parameters were selected based on the range of drug concentrations used in prior vomocytosis studies [16,18,24,46]. This step was followed by time-lapse imaging for 14 hours whilst in drug-containing media.

Effect of polarizing agents on vomocytosis
For experiments studying the effect of phagocyte polarization on vomocytic frequency, DEX or LPS were added to DCs at 48 hours prior to CN infection. For MFs, IL4/13 or LPS was added to the cells 48 hours prior CN infection. Polarizing agent-containing media was replaced with fresh complete media prior to infection with CN. The infection then proceeded using identical methods as previously outlined.

Time-lapse imaging
Infected cells were kept at 37˚C and 5% CO 2 in the imaging chamber of the BZ-X Fluorescence Microscope (Keyence, Itasca, IL) . Images were taken every 4 minutes for a period of 14 hours and compiled into a single movie file using BZ-X software. Movies were blinded by a third party before manual tracking of CNs and scoring for vomocytosis events by an independent technician. After completing a non-lytic expulsion of at least one CN, the phagocyte was then labeled as a 'vomocyte' regardless of the number of expelled fungal cells or subsequent vomocytosis events. After analysis, the percentage of observed CN-containing phagocytes that had performed a vomocytosis event, or '% vomocytes' was recorded as the vomocytosis rate. For the presentation and discussion of data in this study, '% vomocytes' and 'vomocytosis percentage' are used interchangeably.

DC viability
Dendritic cells were seeded on 24-well plates (75,000 cells/ well) in DC media and incubated at 37˚C and 5% CO 2 . Cells were co-incubated with CNs opsonized using previously mentioned methods. Extracellular CNs were washed with DC media and 14 hours later cell viability was measured using the CyQUANT lactate dehydrogenase (LDH) cytotoxicity assay (Thermo Fisher Scientific) according to manufacturer's instructions. Additional details on the viability assays are provided in the Supplementary Information.

Data and statistical analysis
In each experimental group replicate, 300 randomly selected CN-containing phagocytes from multiple viewing regions were observed and vomocytosis manually quantified. All statistical analyses were performed using GraphPad Prism 9. Data of vomocytosis frequency, timing, and # CN in the different conditions were assessed using a Kruskal-Wallis test corrected for multiple comparisons by false discovery rate (FDR) using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, or unpaired Mann-Whitney test when there were only two groups to compare. Prior to statistical comparison, raw categorical data of vomocytosis occurrence was converted to continuous data by calculation of individual percentage values for each biological replicate. Flow cytometry data was statistically assessed using a one-way ANOVA corrected for multiple comparisons by false discovery rate (FDR) using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli or an unpaired t-test when there were only two groups to compare. All data shown include at least three independent experiments. Original time-lapse movies, upon which manual scoring was performed, are freely available upon request. All column graphs, generated on GraphPad Prism 9, display the individual data points, mean, and standard error mean (SEM). All violin plots, generated on R, visualize the individual event data points, mean (red dot), and box plot containing median and interquartile range. All p-values for significant comparisons are listed in S1 Table.

DC and MF phenotype validation
The phenotypes of the bone-marrow derived DCs and MFs were characterized via flow cytometry to confirm that the cell cultures generated with these protocols were authentic. Cells were derived from robust techniques used in prior literature for generation of murine, bone marrow-derived DCs and MFs via GM-CSF [45] and M-CSF [44] respectively. Moreover, cells derived using the above methods have been widely used in prior studies requiring MFs for vomocytosis experiments [9,18,21], and DCs for Cryptococcal infection experiments [40,[47][48][49]. Here, we validated the growth factor-derived cells using CD11c as a DC marker, and the MF marker, F4/80, via flow cytometry. (Fig 1A) Dendritic cell cultures exhibit low F4/80 and high CD11c levels, while MF cultures display high F4/80 and low CD11c signal. (Fig 1B) Furthermore, RNA sequencing analysis of each cell type display notable clustering and separation by PCA plotting. (Fig 1C) These results confirm that the cultures grown are genuine bone marrow-derived DCs and MFs in line with prior literature [50,51].

Vomocytosis from DCs is independent of CN capsule and infection rates, and is comparable to vomocytosis from MFs
We quantified vomocytic events using time-lapse microscopy and verified their non-lytic nature via cell viability assays. After 2-hour phagocytosis of CN, phagocytic cells were washed for the removal of extracellular CN, and time-lapse imaging experiments were performed during a period of 14 hours (Fig 2A). Vomocytosis events, defined as expulsions of CN from host cell while both remain intact, were observed at both CN:phagocyte ratios (c.p.r.) of 1:1 and 5:1, as shown in representative time lapse images (Fig 2B and 2C). Confocal time-lapse microscopy videos confirmed vomocytosis events by visually verifying instances of increased CN (green) fluorescent intensity upon uncoupling between DC and CN cells over the course of 8 hours (S1 Fig). Overall, vomocytosis occurred at a rate of 17% for MFs infected at a 1:1 c.p.r., 18% for MFs infected at a 5:1 c.p.r., 11% for DCs infected at a 1:1 c.p.r., 13% for DCs infected at a 5:1 c.p.r., and 13% for DCs infected with cap59 CN at a 5:1 c.p.r. (Fig 2D and 2E). The vomocytosis rates of acapsular CN-infected MFs were not shown as this has been investigated previously [10]. For the HK CN control groups, an expulsion rate of only 2% or less was observed for both DC and MF groups infected at 1:1 and 5:1 c.p.r. To confirm that these events were indeed non-lytic, DC viability was tested via an LDH assay. We observed no increased toxicity due to CN infection (S2 Fig). Notably, the vomocytosis rates observed from DCs were not significantly different from those observed from MFs at 1:1 and 5:1 c.p.r. (Fig  2F). For conclusive visual confirmation of vomocytosis, high resolution confocal microscopy was used to observe two fixed DC cells mid-vomocytosis at 6 hours after infection with CN (Fig 2G).

Disruption of phagolysosomal maturation inhibits vomocytosis
Next, DCs were treated with drugs previously documented to affect phagolysosomal maturation, and vomocytosis from MFs. Namely, CQ, CYT, and BFA were used for these experiments (Fig 3A). After assessing potential toxicity to DCs and CNs at the desired concentrations (S2 Fig), the individual drugs were added to wells containing CN-infected DCs at a 5:1 c.p.r., just prior time-lapse imaging. All drugs (at the selected concentrations) reduced vomocytosis rates (Fig 3B), although CYT and CQ treatments did not show significance in this reduction. For DCs, we observed vomocytosis rates of 4%, 5%, 6%, and 6% for CQ, CYT hi, CYT lo, and BFA respectively. We confirmed that these inhibited rates were not due to the impact of the drugs on immune cell and CN cell viability (S3 Fig). Furthermore, in CNinfected DC cultures treated with these drugs, there were no observed increases in toxicity to extracellular or intracellular CNs (S4 Fig).

Treatment of MFs and DCs with polarization agents alters their immune phenotype
Prior to testing the effect of immune state on vomocytosis, verification of the immunophenotype of MFs and DCs was performed (Fig 4A). In these experiments, the immature MF (iMF; Control), activated (LPS), anti-inflammatory (IL4/13), and tolerized then challenged (IL4/13 + LPS) conditions were probed (Fig 4B). The LPS-treated group displayed significantly higher inflammatory marker expression of CD38 and iNOS compared to the immature, untreated group. Interestingly, the tolerogenic marker Arg1 was also increased on the LPS-treated MFs; this finding may be due to an inhibitory feedback loop following earlier inflammation in this group. On the other hand, the IL4/13-treated group showed no significant differences for CD38 and iNOS, compared to the immature group, whilst exhibiting higher Arg1 expression than the untreated and LPS groups. Additionally, the tolerogenic group challenged with LPS displayed similarly high levels of Arg1. Additionally, this group had similar expression of CD38 and iNOS markers to the LPS-treated cells.
For DCs, the inflammatory markers MHCII, CD80, and CD86 were analyzed via flow cytometry on CD11c+ gated cells. The immature DC (iDC; Control), activated (LPS), and (D-F) Graphs of vomocytosis rates of MFs and DCs are shown at 1:1 and 5:1 c.p.r., compared to a HK CN control and acapsular cap59 CN (N�4 for each condition, statistical analysis performed using a Kruskal-Wallis test corrected for multiple comparisons by FDR using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Raw tolerized (DEX) conditions were analyzed. Additionally, an added DEX-treated DC group was challenged with LPS (DEX + LPS) for an additional 48 hours to test resistance to inflammatory activation. The LPS-treated group displayed significantly higher MHCII, CD80, and CD86 expression compared to the immature untreated group (Fig 4C). Meanwhile, the DEX group categorical data of vomocytosis occurrence was converted to continuous data by calculation of individual percentage values for each biological replicate). (G) Confocal images showing instances of DCs (DiD, red) expelling CN (CFW, green). The events are visualized via both a combined Z stack and a 3D volume rendering.
https://doi.org/10.1371/journal.pone.0280692.g002  displayed no significant difference to the immature group on the basis of these inflammatory markers. Furthermore, when tested with LPS after DEX treatment, these tolerogenic DCs displayed significantly lower activation of MHCII and CD86 expressions compared to the LPStreated group, indicating resistance toward maturation.

Immune polarization of MFs and DCs does not affect vomocytosis rate
Next, the vomocytosis rates of polarized MFs and DCs were tested (Fig 4D). For MFs, the anti-inflammatory (IL4/13) and pro-inflammatory (LPS) conditions showed significantly higher vomocytosis rates than the heat killed control. However, there were no significant differences in rates between the untreated (18%), anti-inflammatory (13%), or pro-inflammatory (20%) MFs (Fig 4E, left). Similarly, DCs polarized to tolerogenic (DEX) and inflammatory (LPS) phenotypes displayed a higher vomocytosis rate than that of the HK CN group. However, the DC vomocytosis rates of the tolerogenic (12%) and inflammatory (19%) groups were not significantly different to each other or the untreated group (13%) (Fig 4E, right).

Infection ratio, drug treatments, and immune polarization affect vomocytosis kinetics
We used time-lapse videos to measure of exact time of expulsion for each vomocytic event. There was no difference in vomocytosis timing for DCs infected with CN at a 1:1 and 5:1 c.p. r., and no difference compared to DCs infected with the cap59 CN (Fig 5A). For MFs, the 5:1 c.p.r. condition displayed significantly lower average time to expulsion compared to the 1:1 c. p.r. group (Fig 5B). Comparing DCs and MFs, the MF 5:1 c.p.r. condition showed a significantly higher time to expulsion than both the DC 5:1 c.p.r. conditions (Fig 5C). For the drugtreated DC groups, both the CQ and CYT hi treatments performed vomocytosis faster than the DC 5:1 untreated control (Fig 5D). Further, both immune-polarized DCs showed significantly lower time of expulsion than the unpolarized control, with the LPS-treated group having faster expulsion times than the DEX-treated group (Fig 5E). In MFs, the IL4/13-treated condition displayed a longer time to expulsion compared to the control. Additionally, the LPS-treated condition had a significantly faster time of expulsion than the IL4/13-treated group (Fig 5F).

Different treatments affect number of CN per vomosome
In addition to characterizing the vomocytosis rates and timing of different conditions, the number of CN located in the phagosome before a vomocytosis event, or 'vomosome', was also documented and analyzed. Under different infection ratios, the 5:1 condition had a significantly higher average number of CN per vomosome compared to the 1:1 infection ratio for DCs. (Fig 6A) However, in MFs there was no significant difference in CN per vomosome between the 1:1 and 5:1 infection ratios (Fig 6B). Notably, both the 1:1 and 5:1 c.p.r. MF groups showed higher number of CN located in their vomosomes than both DC 1:1 and DC mAb and human serum. Polarized MFs and DCs were infected with opsonized CN at a 5:1 c.p.r. for 2 hours. Following infection, the phagocytes were washed 5 times and time-lapse imaged for 14 hours. (E) Vomocytosis rates of DCs and MFs at a 5:1 infection ratio are shown for immature (untreated), HK CN, tolerized, and inflammatory states. (N�4, n�11 for each condition, statistical analysis performed using one-way ANOVA corrected for multiple comparisons by FDR using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli or a Kruskal-Wallis test corrected for multiple comparisons by FDR using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Prior to statistical comparison, raw categorical data of vomocytosis occurrence was converted to continuous data by calculation of individual percentage values for each biological replicate).
https://doi.org/10.1371/journal.pone.0280692.g004 5:1 c.p.r. groups (Fig 6C). When analyzing drug-treated conditions, the DC 5:1 c.p.r. groups treated with CQ, CYT hi, or CYT lo had a significantly higher number of CN per vomosome than the control. The BFA-treated DC group showed no significant difference in CN per vomosome compared to the untreated control (Fig 6D). Finally, immune polarized groups were analyzed for differences in number of CN per vomosome. Both DEX-treated and LPStreated DCs were observed to have a higher number of CN located in the phagosome prior to a vomocytosis event compared to the unpolarized control. Additionally, the LPS-treated DC group had a higher CN per vomosome count than the DEX-treated group (Fig 6E). For polarized MFs, IL4/13-treated cells displayed a lower CN per vomosome count to the control. Conversely, the LPS-treated group had a higher CN per vomosome average than the control, as well as IL4/13-treated groups. (Fig 6F).

Discussion
Our novel results show that live CN can perform vomocytosis from DCs, to a similar extent exhibited by both MFs and neutrophils. Here, the occurrence of this phenomenon from DCs was rigorously verified by time-lapse microscopy, confocal imaging, and viability assays. Additionally, the average rate and timing of vomocytosis events from DCs were the same as those from MFs. On the basis of CD11c, F4/80, and RNA expression, the results confidently support the phenotype and purity of DC cultures and MF cultures. Previous literature also corroborates the generation of CD11c+ dendritic cells from non-adherent progenitors treated with GM-CSF [52,53]. Prior CN-related studies have assumed the bone marrow progenitor differentiation methods used to be considered pure for both DCs and MFs [9,18,21,40,[47][48][49]. Using flow cytometry, our results show that DCs have a high CD11c expression. Contrastingly, MFs showed high expression of the F4/80 integrin. It should be noted that in vitroderived and in vivo-isolated DCs and MFs always show expression of both of these markers, but to differential levels in keeping with our observations. Interestingly, the rates of vomocytosis from these two populations of in vitro derived cells are the same, in spite of the differences in F4/80 and CD11c expression. Inferably, in vitro-derived DCs are capable vomocytes. Given our results on DC vomocytosis and those published on MFs and neutrophils, it is likely that some components of this mechanism may be conserved across these cell types derived from common myeloid progenitor cells.
Vomocytosis rates from DCs were observed to have no significant correlation to infection rate or presence of CN capsule. However, DCs infected with cap59 CN contained a higher (although not significant) number of CN per vomosome compared to DCs infected with the wildtype strain (H99). This is not surprising, as the CN capsule is known to be a potent antiphagocytic agent that plays a role in reducing uptake by MFs [54][55][56] and DCs [57]. Additionally, DCs infected at a 5:1 ratio showed higher amount of CN per vomosome than DCs 1:1

PLOS ONE
infected, which may be attributed to the larger number of CN that the DCs encountered during the experiment.
When treated with drugs to modify the phagosome (CQ, BFA), DCs displayed substantially lower rates of vomocytosis (although not in the range of significance for the CQ treatment) compared to the untreated control. If treated with an inhibitor of actin polymerization (CYT lo, CYT hi), a similar, but non-significant decrease is shown in vomocytosis rates. Chloroquine is a weak base that passively diffuses into acidic organelles in the cytoplasm, becomes protonated and prevents maturation and fusion of endosomes and lysosomes [58,59]. Cytochalasin B is an actin polymerization inhibitor that has been demonstrated to induce a release of lysosomal enzymes, modulating lysosomal fusion with phagosomes [60,61]. Lastly, BFA is involved in the inhibition of the vacuolar ATPase in lysosomes [62]. With respect to CQ, our findings of reduced vomocytosis rates (albeit below significance) conflict with previous studies that have successfully demonstrated this drug to increase vomocytosis from J774 MF cell lines [11,18]. However, Yang et al showed that CQ decreases vomocytosis rates from primary murine neutrophils [24]. These discrepancies could be attributed to differences in cell type, as well as source (cell line vs. primary murine cells). Interestingly, the decreased BFA-treated vomocytosis rates we observed from DCs align with observations by Nicola et al (from J774 MF cells), that this drug treatment reduces vomocytosis rates [18]. On the other hand, neutrophils were shown to have no change in vomocytosis rates after BFA treatment [24]. Again, these differences could be due to cell type, as well as cell source. Overall, our findings indicate that vomocytosis from DCs is indeed inhibited by phagosomal alkalinization-by either the weak base CQ or the ATPase inhibitor BFA. Pertaining to CYT treatment, again there is some disagreement within literature on the effect of this drug on vomocytosis. Dragotakes et al observed a decrease in vomocytosis rates of J774 MFs following treatment with either cytochalasin B or D [46]. Conversely, Alvarez et al. saw increased rates of expulsion in the same cell type following cytochalasin D treatment [10]. In neutrophils, cytochalasin D was also seen to increase vomocytosis rates in a study by Yang et al [24]. Our observations for DCs show a decrease in vomocytosis rates, albeit insignificant. These trends align with work published by Dragotakes et al., but are in conflict with the other two studies. It is possible that cytochalasin B may cause lysosomal fusion in the infected DCs and cripple the CN's ability to thrive in the phagosome, although our viability data suggests that intracellular CN are still intact during this treatment. The role of CYT in inhibiting actin polymerization may play a more significant role in limiting the phagocyte's machinery and preventing CN from inducing an expulsion. In investigating average timing of vomocytosis, the CQ-treated and CYT hi-treated vomocytosis events displayed a statistically shorter length of time before expulsion compared to the untreated group. Chloroquine reduces expulsion rates, although slightly below statistical significance, but the CN are released relatively quicker compared to untreated cells. These events may be due to alternate mechanisms such as endosome recycling pathways [63]. Interestingly, within the drug treated groups, the average number of CNs per vomosome were all significantly higher than the untreated group with the exception of the BFA-treated group. It is possible that BFA alkalinization of the phagosome reduces CN dividing ability, as CN has been observed to replicate faster in acidic environments [19,64].
Treatment of MFs and DCs with polarization agents did not affect the vomocytosis rates compared to the unpolarized control. This result is contrary to some documented literature for MFs, as Gilbert et al. found that inflammatory-polarized MFs (via ERK5 inhibition) displayed a higher rate of expulsion than untreated MFs, shown in both primary human MFs and J774 cells [9]. Other conflicts exist where Voelz et al. showed that anti-inflammatory IL-4 or IL-13 treated MFs perform a lower rate of vomocytosis compared to an untreated control, also in both primary human MFs and J774 cells [23]. However, the same study showed that the pro-inflammatory IFN-γ, TNF-α, or IL-17 treated human and J774 MFs did not have a significantly different vomocytosis rate than unpolarized MFs, which aligns with our observations. Interestingly, a recent study by Zhang et al. found that primary murine MFs treated with inflammatory extracellular vesicles displayed lower vomocytosis rates [65]-this result contradicts both this study and previous studies. Overall, investigation of the effect of immune phenotype on vomocytosis has been inconclusive. This may be attributed to differences in cell types, polarization procedures, phenotype validation and vomocytosis observation methods. Our results suggest that immune polarization is not a significant influence on vomocytosis rates from MFs or DCs. However, there was a significant difference in the average time for vomocytosis occurrence for these groups. Cryptococcal cells performed vomocytosis in a significantly shorter time from LPS-treated DCs compared to DEX-treated DCs. Similarly, the average non-lytic exocytosis time of CNs in LPS-treated MFs was significantly longer in time length compared to that of IL4/13-treated MFs. This observation is likely due to the differences in molecular and physicochemical characteristics involved in phagolysosomal maturation between activated and tolerogenic phenotypes. For instance, M2 MF have been shown to undergo rapid and profound phagosomal acidification relative to M1 MFs [66]. Moreover, the same study demonstrates that reactive oxygen species (ROS) production is much greater and more sustained in M1 than in M2 phagosomes. Perhaps the mature, ROS and cathepsin-rich phagosome of LPS-treated phagocytes creates an inhospitable environment for CNs, prompting them to escape at a faster rate than in the less harsh anti-inflammatory polarized phagosomes. Additionally, for both DCs and MFs, the LPS-treated groups displayed higher numbers of CN per vomosome compared to the tolerogenic group. The higher number of CN per vomosome and faster ejection from LPS-treated DCs compared to DEX-treated DCs could suggest a direct correlation between number of CN and ejection time-this same trend was observed from DCs treated with CQ and CYT hi.

Conclusions
In summary, this study documents the first recorded observation of DCs performing vomocytosis of CN. Moreover, multiple parameters were tested for their effects on vomocytosis including the infection ratio, presence of CN capsule, treatment with drugs, and polarization of phagocyte immune state. Vomocytosis from DCs is independent of infection ratio, CN capsule, or immune polarization phenotype. However, application of the drug BFA that disrupts the phagolysosome significantly inhibits DC vomocytosis. Interestingly, infection ratio, drug treatments, and immune polarization influence the timing of vomocytosis, as well as the number of CN present in the DC vomosome prior to expulsion. Overall, the capability of DCs to expel CNs following ingestion appears similar to that of MFs based on occurrence rate, timing, and number of CN per vomosome. This finding could help to further elucidate infection dissemination mechanisms in immunocompromised patients, as this phenomenon is clearly conserved between multiple types of phagocytes, including DCs. Finally, more studies are needed to understand the mechanisms involved in vomocytosis from DCs, and moreover determine if there is commonality of processes across all the phagocytic cells that are implicated to perform this behavior. Further, CN is not the only pathogen that has been shown to induce vomocytosis. Therefore, questions on the cohesion of mechanisms across pathogens and phagocytes should be addressed in future investigations.